The goals of this research are 1) to determine how changes in the cytoplamsic mileau modulate ion transport, 2) to elucidate some of the molecular mechanisms of ion transport, and 3) to understand how changes in ion transport rate influence cellular metabolism, physiology and differentiation. The initial approach is to examine how the separate effects of the intracellular proton concentration, (H+)i, the extracellular proton concentration, (H+)o, the phosphate concentration, (Pi), and the membrane potential, Em, and their interactions modulate the operation of the plasma membrane Na/K and Ca pumps of human red blood cells. Defects in pump activity lead to changes in (Ca), (Na), and (K) which alter key cytoplasmic functions and have been implicated in several disease states including renal and heart failure, hypertension, and muscular dystrophy. Alterations in red cell pump activity have been reported in some red cell diseases, including hereditary stomatocytosis and sickle-cell disease. In many cells, physiological challenges affect the unidirectional Na or Ca influx including mitogen stimulation of cell growth, generally increase Na/K or Ca pump activity. Thus, how the cell senses the increased influx rate needs to be determined as well as how the cell modulates the pump rate in the absence of changes of (Na)i and (Ca)i. Alterations in (H+), Em, and (Pi) are known to occur and if they are not the primary means of cellular regulation they must complement or hinder other regulatory mechanisms. The separate effects of (H+)i and (H+)o will be examined because a) changes in (H+)i and (H+)o require different responses from the cell and b) the effect of H+ on the transport mechanism is often obscured when both (H+)i and (H+)o are varied, especially since recent models for these pumps suggest that H+o (or H+i) is a (alternative) substrate. The combined information from the kinetic effects and from effects due to alterations in free energy will clarify the type of regulation and provide constraints on the models of coupling between ATP hydrolysis, ion movement and charge movement. The techniques and insights developed for the red cell will then be tested, refined and altered to understand the regulatory influence of changes in (H+), (Pi), and Em on the ion transport activity of more complex cells.